Promising primers for detection of phytoplasma causing coconut lethal yellowing disease in Mozambique

Coconut is one of the main cash crop in Mozambique, which occupied the second position after Tanzania in coconut production in Africa. Coconut production was drastically affected by the occurrence of a devastating Coconut Lethal Yellowing Disease (CLYD) epidemics, which reduced significantly the coconut yields. CLYD symptoms triggered upon phytoplasma infection, i.e. premature fruit dropping, necrosis of the inflorescence and progressive yellowing of the leaves, are used to identify infected trees. However, the diagnostic based uniquely on symptoms is not conclusive to confirm infection, and needs to be confirmed by molecular methods. In this study, three previously described reference primers for phytoplasma detection were tested on infected samples collected in Mozambique. Since those primers gave incongruent results, 20 new primer pairs targeting the 16S rDNA region, were newly designed. To evaluate their performance in detecting coconut infecting phytoplasma, 108 samples were tested and selected positive samples confirmed by sequencing. Our results showed a new primer pair more accurate and reliable compared to the reference pairs for CLYD detection in Mozambique. Moreover, the new primer pair was able to detect a new putative phytoplasma variant in Mozambique. Therefore, this study makes an important contribution to CLYD phytoplasma molecular diagnostics and its causative agent, giving insights that may be applied to the study of CLYD phytoplasma infection systems.